This grant proposal seeks to evaluate the ability of a unique, non-cytopathic Kunjin virus (KUN) replicon-based vaccine delivery system to induce long-term protective cytotoxic (CTL) responses. The main feature of the system is the ability of the KUN replicon RNA to support its own replication in the cell cytoplasm that results in enhanced levels of immunogen expression. The functional replicon RNAs encoding immunogens can be delivered in vivo by plasmid DNA and/or by Virus-like particles (VLPs) containing replicon RNA packaged into the KUN envelope. HIV gag and an HIV HLA-A2 CTL polyepitope (polytope) will be used as model HIV immunogens and will be delivered by both plasmid DNA and VLP-based KUN replicon vaccines. The in vitro expression of immunogens by these vaccines will first be monitored by Western and Northern Blots and their immunogenicity will be tested in conventional and HLA-A2 transgenic mouse models. The CTL precursor frequency (by ELISPOT analysis) and protection (in a recombinant vaccinia challenge model) induced by KUN vaccines will be measured over extended periods post vaccination and compared with conventional vectors bearing the same immunogens. The grant proposal also intends to develop an improved packaging system for production of KUN-HIV replicon VLPs with high titers. If KUN vectored vaccines prove to be highly immunogenic in mice, the investigators intend to construct and characterize KUN replicon vectors and/or VLPs expressing SIV gag and HIV tat and rev genes for future SHIV challenge experiments in macaques. KUN vaccine vectors generate self-replicating mRNA without inducing cytopathic effects (cell death) and produce high levels of immunogen for extended periods. The investigators postulate that these novel features will result in efficient induction of long lasting CTL responses and that KUN-based replicons will therefore prove valuable in the design of HIV vaccines.